Journal of Invertebrate Pathology 106 (2011) 92–109
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Journal of Invertebrate Pathology
journal homepage: www.elsevier.com/locate/jip
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Diseases of Nephrops and Metanephrops: A review
Grant D. Stentiford a,⇑, Douglas M. Neil b
a
European Community Reference Laboratory for Crustacean Diseases, Centre for Environment, Fisheries and Aquaculture Science (Cefas), Barrack Road, Weymouth, Dorset
DT4 8UB, United Kingdom
b
Department of Environmental and Evolutionary Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, United Kingdom
a r t i c l e
i n f o
Keywords:
Hematodinium
Myospora
Disease
Infection
Decapod
Crustacean
Fishery
Pathogenesis
a b s t r a c t
Nephrops and Metanephrops are commercially exploited genera within the family Nephropidae (clawed
lobsters). Commercial fisheries for each genus exist in the Northern and Southern Hemispheres and utilise trawling or trapping for capture. Despite a relative lack of dedicated disease surveys on lobsters from
these fisheries, several important symbionts and pathogens have been described. The most significant
known pathogen of Metanephrops (challengeri) is a microsporidian parasite (Myospora metanephrops)
which causes destruction of the skeletal and heart muscles of infected lobsters while the most significant
known pathogen of Nephrops (norvegicus) is a dinoflagellate parasite assigned to the genus Hematodinium.
This parasite has been responsible for an ongoing epidemic in fished populations of N. norvegicus in
Northern Europe since at least the early 1980s and since then extensive studies on its life history and
pathogenesis have occurred. Despite these research efforts significant gaps exist in our knowledge of
the effects of parasites such as Hematodinium on the fished and non-fished portions of Nephrops populations and on the effect of fishery practices on the spread of infection. Furthermore, little is known about
the effect of this (and other) pathogens on cohort survivability and the likelihood that early life stages
will be effectively recruited to the fishery. This review summarises the available literature on diseases
of these two lobster genera and provides an assessment of future research needs in this discipline.
Crown Copyright Ó 2010 Published by Elsevier Inc. All rights reserved.
Contents
1.
2.
3.
The Clawed lobsters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1.
Genus Metanephrops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2.
Genus Nephrops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Diseases of Metanephrops. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Diseases of Nephrops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.
Epizoic symbionts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.1.
Symbion pandora . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.2.
Epizoic barnacles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.3.
Epizoic Foraminifera. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.
Pathogens and parasites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.1.
Bacterial shell disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.2.
Systemic Mesanophrys-like ciliate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.3.
Porospora nephropis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.4.
Stichocotyle nephropis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.5.
Histriobdella homari . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.6.
Copepod and isopod infestations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.
Conditions induced by capture, handling and in culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.1.
Idiopathic muscle necrosis and systemic bacteraemia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.2.
Black spot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.3.
Diseases of N. norvegicus during culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.4.
Light damage to eyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
⇑ Corresponding author. Fax: +44 1305206601.
E-mail address: [email protected] (G.D. Stentiford).
0022-2011/$ - see front matter Crown Copyright Ó 2010 Published by Elsevier Inc. All rights reserved.
doi:10.1016/j.jip.2010.09.017
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4.
5.
93
Hematodinium sp. – a model disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
4.1.
Hematodinium sp. as an emergent pathogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
4.2.
Taxonomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
4.3.
Hematodinium diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
4.4.
Current status of infection prevalence monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
4.5.
Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
4.6.
Pathology and its effect on commercial products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
4.7.
Hematodinium changes host behaviour . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
4.8.
Hematodinium epizootiology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
4.9.
Direct and indirect mortality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Conclusions and future directions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
1. The Clawed lobsters
Four families comprise the crustacean group refered to as the
lobsters; Nephropidae (clawed lobsters), Palinuridae (spiny lobsters), Synaxidae (furry lobsters) and the Scyllaridae (Slipper lobsters). Despite the numerous species that comprise these
families, only relatively few are commercially important since
most are small, do not aggregate or live in deep water – life history
traits that make their expolitation less feasible (Dow, 1980; Holthuis, 1991). This review covers diseases of the clawed lobsters
comprising the Nephropidae, with Palinurid lobsters covered elsewhere within this volume. The Family Nephropidae (Dana, 1852)
includes the sub-family Nephropinae (Dana, 1852) which contains
a number of commercially exploited genera including Homarus,
Nephrops and Metanephrops. Two other genera, Eunephrops and
Thymopides are also classified within the Nephropinae. Diseases
affecting lobsters of the genus Homarus are covered in a separate
review within this volume. Furthermore, lobsters within the genera Eunephrops and Thymopides are deep-water species with limited distribution in the Western Atlantic (Eunephrops bairdii,
Eunephrops cadensi and Eunephrops manningi) and southern Indian
Ocean (Thymopides grobovi, Thymopides laurentae), respectively.
Whilst lobsters within these genera are large enough for exploitation and thus are of potential interest for fishery exploitation, lack
of detailed knowledge on their distribution and life history coupled
with their deep water habit (up to 1200 m) preclude a profitable
fishery at present (Burukovsky and Averin, 1977; Holthuis, 1991;
Poupin, 1993; Diaz et al., 2003 Segonzac and MacPherson, 2003).
In light of the current review, it is perhaps not surprising that no
published information exists on the pathogen fauna of these lobster genera. With the exception of the Homarids, lobsters within
the genera Nephrops and Metanephrops are the only commercially
exploited members of the Nephropidae.
Zealand (up to 1000 tonnes of M. challengeri per annum) (Fig. 1)
with the global fishery for Metanephrops species often exceeding
1200 tonnes per annum (source: www.fao.org).
1.2. Genus Nephrops
The genus Nephrops contains a single species, the Norway lobster Nephrops norvegicus (originally Cancer norvegicus Linnaeus,
1758). It is found in large commercially exploited populations in
the eastern Atlantic region from Iceland, the Faroes and Norway
in the North of its range to the Atlantic coast of Morocco and the
Mediterranean Sea in the south. Unlike Metanephrops species, N.
norvegicus can be found at depths as shallow as 20 m, with maximum depths of 800 m. It is an important member of the burrowing
marine benthic community on soft sediments, and over the past
50 years it has been the subject of a major fishery in the northeast
Atlantic (Bell et al., 2006). In addition, due to its availability, adaptability to aquarium conditions and convenience for use as a laboratory model, it has been widely studied by the scientific community
and numerous publications describe the feeding ecology (Loo et al.,
1993; Cristo, 1998), reproduction (Farmer, 1974a), moult cycle
(González-Gurriarán et al., 1998), behaviour (Rice and Chapman,
1971; Farmer, 1974b,c; Aréchiga and Atkinson, 1975; Atkinson
and Naylor, 1976; Newland and Chapman, 1989) and fishery for
this species. Considerable advances in our understanding of the life
history of N. norvegicus in the field have assisted with the management of N. norvegicus as a fisheries target (Tuck et al., 1997a,b;
Merella et al., 1998; Sardá, 1998), though as for most other marine
1.1. Genus Metanephrops
The genus Metanephrops is comprised of a number of species,
some of which are the subject of significant commercial fisheries.
All described species are distributed within the Indo-Pacific region,
with identified populations in Madagascar and Africa (Metanephrops mozambicus), Indonesia (Metanephrops andemanicus, Metanephrops velutinus, Metanephrops sibogae), Taiwan (Metanephrops
armatus, Metanephrops formosanus), Hong Kong (Metanephrops
neptunus), Japan (Metanephrops japonicus, Metanephrops thompsoni),
Australia (Metanephrops australensis, Metanephrops challengeri, M.
sibogae, M. velutinus) and New Zealand (M. challengeri). All species
inhabit muddy substrates at depths of between 150 and 800 m and
the majority of species are thought to burrow. The most readily
exploited species (M. andemanicus, M. challengeri, M. japonicus, M.
mozambicus, M. thomsoni) are captured in trawl fisheries following
emergence from their burrows. The largest fishery exists in New
Fig. 1. Metanephrops challengeri captured from the fishery off Southern New
Zealand. Male lobsters (right) and egg-bearing female lobsters (right). Image
courtesy of Dr. Ian Tuck, National Institute of Water and Atmospheric Research
(NIWA), New Zealand.
94
G.D. Stentiford, D.M. Neil / Journal of Invertebrate Pathology 106 (2011) 92–109
fisheries, significant gaps exist with regard to drivers of recruitment and mortality in the fishery. In recent years, N. norvegicus
has become one of the most important shellfish species captured
in the northeast Atlantic, with annual landings of around 60,000
tonnes (source: www.fao.org). The bulk of landings are from trawler capture, with lobsters usually being ‘tailed’ at sea and landed as
‘scampi’. A trapping fishery also exists for the capture of larger animals in sheltered waters or where trawling is not feasible. Trapcaught animals are usually landed and transported live to distant
markets. Such animals fetch a higher unit price but must be of high
vigour to survive the rigors of handling and transportation
(Stentiford and Neil, 2000; Ridgway et al., 2006).
The natural history of N. norvegicus impinges upon its availability to the fishery. Lobsters are captured when present on the surface of the sediment (Farmer, 1974c). Female lobsters spend
much of the winter within their burrows incubating eggs and are
thus largely unavailable to the fishery during these times, causing
a strong predominance of males in the catches (Farmer, 1974b).
The feeding ecology of the female lobster during incubation is
not well understood, though suspension feeding may play a significant role in nutrient supplementation (Loo et al., 1993). Following
spawning, females emerge from the burrow to feed, moult and be
mated by hard-shelled male lobsters (Farmer, 1974a). On the sediment surface, capture by trawlers is further affected by the ability
of lobsters to perform escape swimming. Following disturbance by
the trawling apparatus, lobsters undergo a series of rapid abdominal flexions and extensions (tail flips), which propel the animal
backwards (Newland et al., 1992). Both the speed and endurance
of tail flip swimming have implications for capture by trawl nets.
Once within the net, the retention of captive lobsters is dependent
upon the size of the lobster, the mesh size and the crowding of the
net with other species.
2. Diseases of Metanephrops
Despite a significant fishery or fishery potential for members of
the Metanephrops genus, the combination of their deep-sea habit,
their limited potential for culture (Holthuis, 1991) and their localised sale as fresh or frozen product likely defines the paucity of
published reports on disease in this genus. However, the aggregated distribution that allows their capture in commercially attractive quantities also suggests a rather gregarious habit similar to
that of their sister genera Nephrops. Since dedicated disease surveys of Metanephrops have not been carried out, it is not surprising
that descriptions of viral, bacterial, fungal or protozoan pathogens
are absent from the literature. The literature does however contain
a few reports of crustacean (copepod and isopod) infestations of
Metanephrops collected incidentally through faunal surveys of the
Indo-Pacific region.
Nicothoe analata, a parasitic copepod was described attached to
the gills of specimens of Nephrops (=Metanephrops) sinensis captured from the South China Sea (Kabata, 1966). Later studies by
the same author described a further two species of the same genera (N. brucei and N. simplex); N. brucei infecting Nephrops (=Metanephrops) sagamiensis collected from Japan and from Nephrops
(=Metanephrops) andamanacus collected from South Africa, and N.
simplex infecting Nephrops (=Metanephrops) japonicus collected
from Japan. Furthermore, the host range for N. analata was extended to Nephrops (=Metanephrops) boschmai and Nephrops
(=Metanephrops) andamanacus from Australia and Bali, Nephrops
(=Metanephrops) sagamiensis from Japan, and Nephrops (=Metanephrops) sibogae from Indonesia (Kabata, 1967). The description
of members of this genus infecting Metanephrops follows work
on a similar species, Nicothoe astaci infecting the European lobster
(Homarus gammarus) from Scotland (Audoin and Milne-Edwards,
1826, in Mason, 1959). The genus is considered truly parasitic since
it attaches to the gill lamellae of host lobsters and draws haemolymph via its styliform mandibles that pierce the cuticle (Mason,
1959; Shields et al., 2006). Impact of infection on the host has
not been reported though prevalence is highest in soft-shelled H.
gammarus (Mason, 1959) and parasites are likely shed during
moulting. No data is available on the prevalence of these parasites
in Metanephrops species or on their likely impact on host
populations.
The velvet lobster (M. velutinus) supports a commercial export
fishery in Western Australia (Holthuis, 1991). Additionally, M. velutinus has been captured from the western Indo-Pacific as part of
faunal surveys of the region. As part of these surveys, M. velutinus
has been shown to be type host for the parasitic bopyrid isopod
Pseudione nephropis (Shiino, 1951; Markham, 1999). Pseudione
forms a heterogeneous group of separate sex isopods containing
numerous species infecting a diverse range of decapod hosts
including carideans, thalassinoids, galatheoids, pagurids, lithodids
and nephropids. Infection by Pseudione manifests as a distension
of the affected gill chamber and atrophy of the gill lamellae
(Paradiso et al., 2004) and has been associated with significant
growth reduction and possible physiological castration in other
decapod hosts (Munoz, 1997; Roccatagliata and Lovrich, 1999;
Gonzalez and Acuna, 2004). Based upon the single description of
P. nephropis infection in M. velutinus, no information is available
on the individual or population impact of this parasite.
Perhaps the most significant known pathogen of the genus is
the recently discovered microsporidian Myospora metanephrops
(Stentiford et al., 2010). The parasite was discovered during fishing
surveys of commercially significant stocks of M. challengeri off New
Zealand. Following trawl capture, affected lobsters were clearly
distinguishable from their non-affected counterparts due to lethargy and an apparent alteration in the normal pigmentation and
translucency of the carapace of the cephalothorax, abdomen and
limbs. When observed ventrally, the large abdominal flexor muscles of affected lobsters were clearly visible through the arthrodial
membranes, while muscles extending into the telson blades were
imparted with a distinctive white and opaque appearance which
clearly contrasted the hyperpigmented telson carapace. Histology
reveals a widespread infection of the skeletal and heart musculature in addition to the longitudinal and circular muscles surrounding the hepatopancreatic tubules and the mid- and hind-gut. In
heavily infected lobsters, cysts packed with merogonal and sporogonal stages of the parasite largely replaced muscle fibres and constituent myofibrils of the major abdominal flexor muscles of the
abdomen. Transmission electron microscopy of infected muscle fibres revealed multiple stages of the microsporidian parasite with
development progressing through merogony and sporogony to
the production of mature spores (Fig. 2). All stages were diplokaryotic. Analysis of the SSU rDNA of the microsporidian revealed closest similarity to other muscle infecting microsporidians from
marine crustacean hosts (Stentiford et al., 2010). Interestingly, M.
metanephrops is the first example of a microsporidian parasite in
the clawed lobsters. Further work is now required to investigate
the commercial significance of this parasite, both in terms of population level mortality and effects on the marketability of musculature extracted from infected animals.
3. Diseases of Nephrops
The relatively replete literature concerning symbionts and
pathogens of Nephrops compared to the other genera of clawed
lobster (exclusive of Homarus) is likely testimony to the large numbers of studies on the ecology of this genus. Such ecological studies
when coupled with those describing the fishery for Nephrops across
G.D. Stentiford, D.M. Neil / Journal of Invertebrate Pathology 106 (2011) 92–109
95
Fig. 2. Myospora metanephrops infection in musculature of Metanephrops challengeri (Stentiford et al., 2010). (A) Light micrograph of parasite cysts (white arrows) between
intact myofibrils of abdominal musculature. Bar = 200 lm. (B) Light micrograph of spore stages liberated from a ruptured cyst in the abdominal musculature (arrow).
Bar = 25 lm. (C) Light micrograph of infection of muscle fibres of heart myocardium with associated haemocyte infiltration (arrow). Bar = 100 lm. (D) Light micrograph of
heavily infected heart myocardium (white arrow) with minimal involvement of spongy pericardial cells (black arrow). Bar = 100 lm. (E). Transmission electron micrograph of
abdominal muscle fibre containing early (meront, sporont, sporoblast – black arrow) and late (spore – white arrow) parasite stages. Bar = 2 lm. (F). Transmission electron
micrograph of mature spores showing elongate nature, diplokaryotic nuclei and approximately 11 turns of the polar filament coil. Bar = 0.5 lm.
3.1. Epizoic symbionts
parts of N. norvegicus; these sessile stages produce separate sex,
short-lived and motile stages that allow for re-colonisation of
new hosts (Funch and Kristensen, 1995). The new phylum has similar morphological and life history characteristics to the Entoprocta
and the Ectoprocta and has recently been shown by molecular
analysis of its 18S rRNA sequence to be a sister group to a Rotifera-Acanthocephala clade (Winnepenninckx et al., 1998). Although
its discovery on the mouthparts of N. norvegicus appears remarkable (Morris, 1995) due to the diversity of published works on this
host species, it does highlight a relative lack of basic study on the
symbionts of even our most commercially significant crustacean
species. The presence of similar species infesting Nephrops from
other geographical locations or from other decapod hosts has not
been demonstrated to date.
3.1.1. Symbion pandora
N. norvegicus collected from the Kattegat Straits between Denmark and Sweden are host to S. pandora, the type species of a
new genus within a newly erected phylum, the Cycliophora. The
acoelomate metazoan has sessile stages that inhabit the mouth-
3.1.2. Epizoic barnacles
Balanus crenatus have been described causing epizoic infestations of N. norvgegicus from the Clyde Sea Area, Scotland. Infestations had higher prevalence and were more abundant in larger
animals, presumably related to the longer intermoult period of
its natural range have led to the description of a number of important symbionts, some of which have been shown to have a significant detrimental effect on host populations. The relatively
gregarious nature of Nephrops, its burrowing habit and its existence in large, relatively uniform and sessile populations may contribute to its suitability as a host and may assist with the
transmission of symbionts and pathogens between individuals.
The remainder of this chapter is devoted to a synthetic description
of known symbionts and pathogens of the genus Nephrops. Furthermore, an up to date analysis of the most significant pathogenic
disease of Nephrops (the parasitic dinoflagellate Hematodinium) is
provided.
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older animals and a propensity for smaller animals to inhabit burrows (Barnes and Bagenal, 1951). While epizoic infestations such
as these are unlikely to cause direct harm to host lobsters, their
presence likely relates to decreased moulting frequency and hence
their presence in smaller animals may indicate disruption to normal moulting by pathogens or by environmental perturbations
(Stentiford and Feist, 2005).
3.1.3. Epizoic Foraminifera
An opportunistic study of N. norvegicus captured from the Irish
Sea led to the discovery of epizoic Foraminiferans of the genus
Cyclogyra attached to the pleopods of a single male lobster. The
author notes this as the first example of a Foraminferan infestation
of a decapod crustacean though concedes that lack of study rather
than rarity likely hampered their previous discovery (Farmer,
1977). An interesting observation from this study was the relative
lack of epizoic organisms (including polychaetes, bryzoans, molluscs and cirripedes) infesting N. norvegicus compared to other
decapod crustaceans. The basis of this lack of symbiosis is not
known though may relate to the slender nature of the N. norvegicus
chelipeds, the presence of pincers at the ends of the foremost pairs
of walking legs and the role of the limbs in grooming (Mariappan
et al., 2000; Bell et al., 2006).
3.2. Pathogens and parasites
3.2.1. Bacterial shell disease
Observations of commercial and survey catches of Nephrops report that the majority of populations are comprised of individuals
with clean shells with little fouling and a low incidence of the shell
disease described in several other commercially exploited decapod
crustacean species (Bell et al., 2006). This finding was reinforced in
a study, by Ziino et al. (2002), who assessed shell disease status in
600 N. norvegicus obtained from Italian fish markets. Here, just 1%
of lobsters presented symptoms of shell disease, normally manifested as small erosive and melanised lesions on the chelae. Histopathology of affected regions demonstrated erosion of the cuticle
and infiltration by host haemocytes, often associated with bacterial
colonisation. Pseudomonas spp. and in one case, Enterobacter
agglomerans, both of which have chitinase activity, were isolated
from shell lesions (Ziino et al., 2002). Further analysis of lesions described by these authors suggest an aetiology consistent with
host–host conflict since all lesions were described from the chelae
and appeared as apparent punctures rather than as the extensive
cuticular erosion reported for shell diseased Cancer and Homarus
species (Bayer et al., 1989; Vogan et al., 2001; Smolowitz et al.,
2005). Further studies on comparative aetiology and host susceptibility of shell disease in commercially exploited crustacean species
may assist an improved definition of the condition as predominantly host or pathogen associated and furthermore may help to
elucidate risk factors for its induction in specific fisheries.
3.2.2. Systemic Mesanophrys-like ciliate
Systemic ciliate infections have been reported from a range of
commercially exploited decapod crustaceans, some of which have
gained attention due to their significant ecological and economic
effect. A preliminary observation of a co-infecting Paranophrys-like
ciliate in Nephrops captured from Scottish waters was provided by
Field and Appleton (1996) during their studies on the culture of the
dinoflagellate parasite Hematodinium infecting the same host.
More recently, Small et al. (2005a,b) have described the parasite
in detail as a scuticociliate of the genus Mesanophrys (=Anophrys,
Paranophrys). Paradoxically, while the morphology of the parasite
resembled most closely M. carcini, rDNA sequence data provided
closer affinity with Orchitophyra stellarum, a scuticociliate from
echinoderms. This inconsistency has prevented the naming of the
parasite from Nephrops and Small et al. (2005a) have suggested
that for this reason, data pertaining to the number of somatic kineties may not be a robust enough measure when identifying closely related scuticociliates. As such, they suggest further work to
compare the ITS region sequences for nominal Mesanophrys species
with other known genera in order to resolve this phylogenetic issue. Morado provides a broader discussion on ciliate taxonomy
elsewhere in this volume. Infection by the parasite in Nephrops is
systemic, with proliferation of free stages within the haemal sinuses. A pronounced haemocytopenia accompanies infection, with
an almost complete absence of haemocytes observed in histological section. In vitro culture of the pathogen has shown that it secretes several metalloproteases into the culture medium that are
able to degrade specific components of the host skeletal muscle
when the two are incubated. Specifically, myosin heavy chain, a
critical component of the contractile apparatus in skeletal muscle
is preferentially degraded (Small et al., 2005b). It follows that specific proteases secreted by such pathogens are likely implicated in
the pathogenesis of this and similar diseases of crustaceans caused
by ciliates (Small et al., 2005a,b).
3.2.3. Porospora nephropis
P. nephropis is a gregarine parasite (Apicomplexa) found within
the alimentary canal of N. norvegicus captured from French waters
(Léger and Duboscq, 1915; Tuzet and Ormieres, 1961). Infestations
have also been observed in N. norvegicus captured from the Clyde
Sea Area, Scotland (Field and Appleton, 1995; Stentiford, Neil and
Beevers, pers. obs.), suggesting a wide geographical range for this
parasite of N. norvegicus. A similar species, P. gigantea inhabits a
similar niche within the hindgut of Homarus americanus
(Montreuil, 1954; Théodoridès and Laird, 1970; Boghen, 1978;
Brattey and Campbell, 1985a). The lifecycle of both species alternates between the lobster (in which spore-like gamonocyst stages
develop from characteristic trophonts) and a mollusc, in which
bundles of sporozoites develop, held together by a fragile membrane. Diagnosis of infection cannot be performed externally
though squash or histological preparation of the gut will identify
cyst like structures in the posterior intestine of infected lobsters.
These spherical gamonocysts attach to the cuticular lining of the
gut and may be shed at moult. The long, thread-like trophic stages
inhabit the gut lumen (Bower, 1996) (see Fig. 3). Shields et al.
(2006) state that P. gigantea is perhaps the most common parasite
of Homarid lobsters, with prevalences of between 40% and 100% in
different regions of the host range. Due to its complex multi-host
Fig. 3. Histology of trophic stages of the gregarine parasite Porospora nephropis
inhabiting the midgut lumen of N. norvegicus (arrow). Adjacent hepatopancreatic
(HP) and testicular (T) organs shown. Image courtesy of Dr. Nick Beevers, University
of Glasgow, Scotland.
G.D. Stentiford, D.M. Neil / Journal of Invertebrate Pathology 106 (2011) 92–109
lifecycle, the availability of suitable molluscan intermediate hosts
likely implicates its presence in a particular fishery (Van Engel
et al., 1986). The role of Porospora as a disease agent in lobster populations is not well understood and no studies have been carried
out on the role of this parasite either as a driver of host mortality
or as to a subtler role as a modulator of host physiology or
reproduction.
3.2.4. Stichocotyle nephropis
N. norvegicus from the Firth of Clyde, Scotland are host to the
parasitic trematode S. nephropis. The parasite was first described
by Cunningham (1887) who discovered cyst-like protuberances
in the hindgut that upon further inspection were seen to contain
a novel genus of Trematode. Following excystment, S. nephropis
were observed to vary in length from less than 1 mm to 8 mm.
The ventral surface of each worm contains a series of suckers along
the median line, with the number varying according to the size and
presumably age of the worm. Cunningham (1887) notes that more
than one worm may be found within each cyst and that the wall of
the cyst is cellular in nature and likely a ‘pathological product’ of
the intestinal tissue of the host. Interestingly, the worms were
discovered in an excysted (but live) state in recently dead animals
and large numbers (up to 40) were found within a single host. In
this early work, the prevalence of infection was estimated to be
up to 25% though this varied between sampling sites. While
Cunningham (1887) only described the larval form within N. norvegicus, later work by Odhner (1898) described what were believed
to be the adult stages of S. nephropis in bile ducts and the spiral
valve of the thornback ray, Raja clavata from Swedish waters. MacKenzie (1963) also reported on the prevalence of the adult parasite
within R. clavata from Scottish waters. MacKenzie (1963) reports
infection prevalence of between 1% and 48% in N. norvegicus collected from different locations within the Scottish fishery and his
data is suggestive of a higher prevalence and intensity of infestation in larger animals captured on the west coast. In this study,
of the 14 R. clavata assessed, only one male was shown to be
infected with adult stages of S. nephropis. A further study of
S. nephropis infestation of N. norvegicus, from the Scottish and
English fishery, was carried out by Symonds (1972). Symonds
reports on an increase in prevalence with size in both sexes and almost without exception, the parasite was absent from animals
with a carapace length of less than 30 mm. Interestingly Symonds
(1972) also reports a reduced prevalence of infection when compared to original surveys by Cunningham (1887), stating that this
may indicate that this parasite is less common in British waters
than reported in the late 19th century. Whether evidence for reduced prevalence of larval S. nephropis in N. norvegicus acts as a
surrogate for data pertaining to reductions in stock abundance of
European elasmobranch species such as R. clavata (Rogers and Ellis,
2000) requires further assessment but does highlight the potential
importance of multi-host crustacean parasites as indicators of
community integrity (Stentiford and Feist, 2005).
3.2.5. Histriobdella homari
H. homari, a eunicid polychaete worm has been described infecting the branchial chamber and egg mass of N. norvgegicus from
British waters. The symbiont was first described by Van Beneden
(1858) and subsequently studied by Briggs et al. (1997) in Irish
Sea stocks. H. homari has also been described infesting the gill
chamber and eggs of H. americanus from US and Canadian waters
and H. gammarus from Europe (Sund, 1914; Bruce et al., 1963;
Uzmann, 1967; Brattey and Campbell, 1985b; Lerch and Uglem,
1996; Shields et al., 2006). Early descriptions of infestations suggested H. homari as an egg predator associated with poor larval production in European lobster hatcheries (Sund, 1914) though later
studies by Lerch and Uglem (1996) demonstrated no correlation
97
between hatching success and high-intensity infestation. Shields
et al. (2006) classify the likely association between H. homari and
its lobster host as symbiotic due to the grazing habit (on bacteria
and protozoans associated with the egg mass), its high prevalence
(up to 100% at some sites – Uzmann, 1967), high-intensity (Brattey
and Campbell, 1985a,b) and aforementioned absence of observed
effect on egg mortality.
Infection prevalence and intensity in N. norvegicus appears to be
less than that reported in populations of H. americanus. In a survey
of Irish Sea N. norvegicus only isolated examples were discovered
attached to the pleopod setae of male specimens (Briggs et al.,
1997). Personal accounts of those working on N. norvegicus in Scottish waters (see Briggs et al., 1997) hint at the apparent rare and
potential incidental nature of infestation within N. norvegicus, at
least within those populations regularly assessed.
3.2.6. Copepod and isopod infestations
Copepod and isopod infections of N. norvegicus have been rarely
reported in the literature. Thomson (1896) describes an Anchorellalike copepod from the vas derefens of N. norvegicus captured in
Scotland. Affected individuals displayed a large and distinctive
swelling on the wall of the vas deferens that caused distension of
the thoracic cavity. The swelling was elicited by a large (up to
8 mm) female copepod accompanied by a morphologically dissimilar (5 mm) male. Subsequent examinations of N. norvegicus by
Thomson (1896) defined the apparent prevalence of infection as
less than 1% though no comprehensive surveys were carried out
on lobsters obtained from different fishing grounds. Thomson
(1896) acknowledged that at the time of writing, little was known
about the life history of Anchorella-like copepods nor how they
came to infect male hosts. No further descriptions of this parasite
are apparent in the literature and as such, the intimate endoparasitic nature of this copepod infection and its close association with
the male reproductive system of N. norvegicus remains an interesting model for furthering our understanding of copepod infestations
in higher crustaceans.
One intriguing report of infestation of N. norvegicus by parasitic
isopods exists within the current literature. During a fishing survey
of the eastern Mediterranean, Atesß et al. (2006) discovered two
parasitic isopod species belonging to the family Cymothoidae associated with the egg clutch of gravid female N. norvegicus. One specimen was identified as a male Ceratothoa italica (normally found
infecting the buccal cavity of Mediterranean teleosts) while the
other, a provisional male of Livoneca pomatomi (occasionally found
infecting the branchial cavity of certain bathypelagic teleosts).
While cymothoid isopods have been described associated with a
number of non-teleost hosts previously, the authors note that in
this case, there is some degree of uncertainty as to whether these
parasites form true infections or whether they are accidental associations. In certain cases, such as in Telotha henselii infection of
Macrobrachium brasiliense, Macrobrachium borelli and Pseudopalaemon bouveri, studies have suggested that the association is not
accidental but rather that young males of this species may utilise
shrimp as intermediate hosts (Lemos de Castro, 1985; Grassini,
1994). Whether such association is true for the isopod infestation
of N. norvegicus remains to be shown.
3.3. Conditions induced by capture, handling and in culture
3.3.1. Idiopathic muscle necrosis and systemic bacteraemia
A rapid onset abdominal muscle necrosis (termed idiopathic
muscle necrosis, IMN) has been identified in Scottish N. norvegicus
immediately following trawl capture (Stentiford and Neil, 2000).
IMN has led to economic losses, particularly in the live market
where affected animals succumb to initial signs of the condition
within 4 h of capture, with rapid progression through the following
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24 h. Affected individuals show a characteristic whitening of the
abdominal muscle, firstly as individual muscle fibres, followed by
progression of the lesion to adjacent muscle fibre groups, abdominal segments and eventually the whole abdomen. At this advanced stage, the commodity product (extruded abdominal
muscle) has a significantly altered texture prior to and following
cooking (Stentiford and Neil, pers. obs). Histopathological and
ultrastructural studies of the condition reveal a progressive necrosis of affected muscle fibres, with loss of sarcomeric structure and
moderate influx by host haemocytes. Ultrastructurally, sarcomeric
disruption is confirmed by first a loss of structure in the region of
the Z-line followed by separation and necrosis of individual myofibrils. Necrosis appears to progress longitudinally through the myofibres, leading eventually to a homogenous matrix of cellular
debris and necrotic cell products (Stentiford and Neil, 2000). The
pathology, coupled with the loss of contractile proteins demonstrated using SDS–PAGE causes a loss of normal functioning of
the abdomen and the characteristic ‘tail flip’ cannot be induced
(Stentiford and Neil, 2000).
While the original description of the condition did not elucidate
an aetiology (hence the designation as ‘idiopathic’), the progression
and pathogenesis of IMN has been studied more recently by
Ridgway et al. (2006, 2007). Using a multivariate approach measuring physiological, endocrinological, immunological, microbiological
and pathological indicators, Ridgway et al. (2006) demonstrated
that in the crucial period of air exposure following trawl capture,
N. norvegicus experienced large disruptions in physiological profile
(increases in haemolymph L-lactate, crustacean hyperglycaemic
hormone and carbohydrates and fluctuations in haemolymph pH).
Furthermore, during this period, the immune competence of
lobsters is impaired (indicated by reductions in circulating haemocyte titre and phenoloxidase levels). During the period immediately
following capture, potential for systemic microbial infection increases (assisted by integumental damage), demonstrated by significantly increased counts of opportunistic bacterial species
(Ridgway et al., 2006). Recently, Ridgway et al. (2007) have developed this principle by suggesting that IMN in N. norvegicus may occur as a two-stage pathogenic process, with an original lesion
induced by acidosis from continuous rapid tail flipping during the
capture process and a subsequent development of the condition
as bacteraemia in immunosuppressed lobsters exposed to further
stressors during the post-capture period. The IMN model in
N. norvegicus serves as an excellent example of disease induction
via commercial production processes and highlights how directed
and applied scientific research can inform on improved practice
and ultimately to assist in reducing economic impact of emergent
conditions. It has been suggested by Ridgway et al. (2006) data of
this type may be used to generate an internationally accepted Code
of Practice for the capture, handling and transport of commercially
exploited decapod crustaceans. Furthermore, it may be used to inform on best practice for disease limitation in aquaculture settings.
These issues are discussed more fully by Fotedar and Evans elsewhere in this volume.
3.3.2. Black spot
While not considered to be a disease, ‘blackspot’ development
in N. norvegicus is worthy of mention since it has been responsible
for significant losses during the post-capture period when lobsters
are stored on ice. Blackspot occurs as a result of polyphenol oxidase
(PPO) oxidising diphenols to quinones that further undergo autooxidation and polymerisation to form dark pigments. Yan et al.
(1989) observed a linear relationship between PPO and the rate
of colour development in homogenates of N. norvegicus. Bartolo
and Birk (1998) investigated factors that influence the degree of
blackspot development in N. norvegicus. Due to the role of PPO in
sclerotization of the exoskeleton following moulting, several stud-
ies on other decapod species have demonstrated how blackspot
formation appears to be related to the moult stage of the host, with
higher levels of PPO in decapods preparing to moult and in cast-off
shells (Ogawa et al., 1983, 1984a,b). Further, Bartolo and Birk
(1998) report that PPO activity peaked during the months coincident with the major moulting season in N. norvegicus. However,
they also demonstrated that initial levels of PPO were not correlated with susceptibility to blackspot development post-capture;
rather that changes in enzymatic activity during the post-capture
and storage period may indicate likelihood of blackspot development. Their conclusion that the condition can be induced by ‘traumatic’ events such as capture and rough handling is further
testimony to a requirement for good handling practice in fishers
and those with market interests in this product. Industry experience and recent research have concluded that reducing agents
based on sulphites are the most effective and practical control
agents for melanosis in N. norvegicus, when compared to a large
variety of chemical alternatives. However, due to their possible adverse effects on humans, EU regulations restrict the concentrations
of sulphites to a maximum of 10 mg of SO2 kg1. Recent work has
shown that alternative treatments based on 4-Hexylresorcinol,
which inhibits PPO activity, can be equally effective (MartınezAlvareza et al., 2007).
3.3.3. Diseases of N. norvegicus during culture
Since sustained attempts to grow N. norvegicus by aquaculture
have not occurred, few reports are available on their susceptibility
to disease when held in captivity. However, in a study of marine
crustaceans with potential for aquaculture, Anderson and Conroy
(1968) report on heavy infestations by ciliates of the genus Zoothamnium on the body surfaces of live N. norvegicus larvae hatched
in the laboratory. They report that heavy infestations led to death
as a result of ‘trauma and interference to respiration’. A general
observation regarding clawed lobsters, in particular with regard
to attempts to raise them in closed culture systems is the apparent
absence of viral pathogens in this group. Viral pathogens have
caused massive economic impact on the culture of tropical penaeids (see the paper by Lightner in this volume) and recently the first
virus has been described in spiny lobsters of the Palinuridae
(Shields and Behringer, 2004 – see also the paper by Shields in this
volume). Whether the absence of reports of viral pathogens in
Nephropid lobsters reflects a true resistance of this group to
viruses or whether insufficient field surveys have been carried
out (of larvae, juveniles and adults) to describe pathogens can only
be speculated. However, differential resistance of specific crustacean groups to economically significant viral pathogens may provide a fruitful research area and may directly benefit the global
requirement for sustainable development of crustacean aquaculture (see Stentiford et al., 2009).
3.3.4. Light damage to eyes
The eyes of N. norvegicus are large, well pigmented and approximately kidney-shaped (the basis of their generic name ‘Nephrops’
meaning ‘kidney-eye’). Several studies on the behaviour of this species have shown it to spend the majority of its time within its burrow, with emergent forays to feed and mate (Chapman and Howard,
1979; Chapman, 1980; Bell et al., 2006). They have a well-described
diurnal activity pattern that coincides with peak catch rates via
trawling and this varies according to the depth of water of resident
populations (Chapman and Rice, 1971; Chapman and Howard,
1979). While light (and the eye) is important for maintenance of
this pattern, several behavioural studies have shown that other factors (such as food availability and even innate endogenous
rhythms) also play a role (see Bell et al., 2006). Shields et al.
(2006) have recently summarised the pathological effects of light
on eye of N. norvegicus. Light damage to the eyes of N. norvegicus
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was first observed by Loew (1976). Subsequent studies by Shelton
et al. (1986a,b) and Gaten (1988) followed the pathogenesis of
the condition that proceeded to a loss of retinula cells in the ommatidia and haemocytic infiltration of necrotic regions. In later field
studies of tagged and released lobsters with light damaged eyes,
Chapman et al. (2000) demonstrated that once damaged, the eyes
did not recover their ability for light adaptation. However, light
damage did not appear to influence mortality since recaptures occurred up to several years following release.
4. Hematodinium sp. – a model disease
Of the known pathogens and parasites described from N. norvegicus, by far the most significant in terms of ecological and economic impact is the dinoflagellate parasite Hematodinium sp.
Disease associated with this pathogen had been reported anecdotally in Scotland since the early 1980s, during which period it was
termed ‘post moult syndrome’ due to the coincident appearance
of affected lobsters during the main moulting period for N. norvegicus. However, it was not until the early 1990s that ‘post moult syndrome’ was shown to be associated with a dinoflagellate parasite
of the genus Hematodinium, previously only known to infect crabs
(Field et al., 1992). Since this description, Hematodinium and Hematodinium-like species have been discovered in an increasing array
of decapod and non-decapod crustacean hosts (see elsewhere
within this book), many of which are the subject of commercially
important fisheries. In a recent review of this pathogen group,
Stentiford and Shields (2005) suggest them to be one of the most
significant (known) pathogens of wild marine crustaceans. Since
the majority of studies carried out on Hematodinium have been
in commercially exploited hosts, there is high likelihood that this
parasite group is distributed widely amongst decapod (and some
non-decapod) hosts. The section that follows is devoted to a
description of the disease caused by Hematodinium sp. in N.
norvegicus.
4.1. Hematodinium sp. as an emergent pathogen
Routine examination of N. norvegicus from the west of Scotland
fishery in the early 1980s led to discovery of a low prevalence on
lobsters that displayed an opaque but apparently hyperpigmented
carapace, milky-white haemolymph and a general moribund status
following capture. The condition, termed ‘post moult syndrome’
was initially thought to be due to a hyperplastic increase in host
haemocytes associated with the concurrent moult season of N. norvegicus in the region. However, by the late 1980s the increased
prevalence and poor quality of animals showing these symptoms
began to evoke comment from fishermen and processors and a
full-scale survey was launched (Field et al., 1992). Detailed examination of affected lobsters revealed that they were infected by
masses of non-motile protistan parasites that resembled dinoflagellates of the Order Syndiniales, most similar to the parasite Hematodinium perezi, described by Chatton and Poisson (1931) from
European portunid crabs (Field et al., 1992). The parasite also
resembled Hematodinium-like dinoflagellates that had previously
been described from the crabs Callinectes sapidus (Newman and
Johnson, 1975), Cancer irroratus, Cancer borealis, Ovalipes ocellatus
(Maclean and Ruddell, 1978), Necora puber (Wilhelm and Boulo,
1988), Cancer pagurus (Latrouite et al., 1988), Chionoecetes bairdi
and Chionoecetes opilio (Meyers et al., 1987, 1990; Eaton et al.,
1991). The discovery of Hematodinium sp. in N. norvegicus was
highly significant since it marked the first description of a Hematodinium-like pathogen in Nephropid lobsters and it appeared to
show similar pathological and epizootiological features to those
previously described in infections of commercially exploited crab
99
species. In the subsequent decade, a significant work program to
describe and diagnose the pathogen and to investigate its effect
on the life history traits and population structure of its host
followed.
4.2. Taxonomy
Despite its commercial significance, the taxonomy of Hematodinium spp. and the Hematodinium-like dinoflagellates remains confused with most isolates (including that from N. norvegicus) still
only named generically. Despite significant advances in molecular
diagnostic capabilities (see below), the description of isolates of
Hematodinium spp. as specific entities in their respective hosts
has been hampered by a failure to compare ‘new’ isolates to the
type species (H. perezi) from type hosts (Carcinus maenas or Liocarcinus depurator) collected from the type location (English Channel,
off France) as detailed by Chatton and Poisson (1931). The paucity
of studies of the type host has largely been attributed to the very
low prevalence of H. perezi in European portunid crabs (see
Stentiford and Shields, 2005). However, recent studies have shown
that while infection prevalence in C. maenas may in fact be rather
low (less than 1%), with infected crabs also displaying low-level
infections (Stentiford and Feist, 2005), infection prevalence in L.
depurator may be significantly higher, with animals displaying
pathologies similar to those associated with Hematodinium sp.
infection in hosts such as N. norvegicus. Recent unpublished work
has reported H. perezi in L. depurator at sites in the English Channel
(between France and the United Kingdom) at prevalences of up to
25% (Stentiford, G.D., pers. obs.). The pathological outcome of
infection appears similar to all other published accounts though
the ultrastructure of known isolates is somewhat varied (Fig. 4)
compared to the type species. Stentiford and Shields (2005) state
that the description of H. perezi as type species in C. maenas and
L. depurator may have been unfortuitous for taxonomic studies of
other isolates due to the difficulty in obtaining type material. However, it appears that this may have been an oversimplification since
infection prevalence and severity appears to differ between the
two ‘type’ hosts. Work is currently underway to compare Hematodinium sp. isolates from N. norvegicus and commercial crab species
with H. perezi from L. depurator collected from the English Channel
(Small, H.J., pers. comm.). Further, due to the apparent differences
in host-pathogen relationship observed in C. maenas and L. depurator, it is possible that the original ‘type’ description from these two
hosts may need to be modified to describe two discrete species
with differing pathological outcomes. Further efforts are now
required to gather appropriate material from H. perezi-infected C.
maenas in order to provide taxonomic clarity in the group. Only
when this occurs can we investigate the global epidemiology of
this important disease and calculate risk factors for its transfer to
naïve geographical locations and hosts.
4.3. Hematodinium diagnosis
Several tools are available for diagnosis of Hematodinium infections in N. norvegicus. Intriguingly, heavily-parasitised animals can
be recognised externally via an increased opacity and pronounced
hyperpigmentation of the carapace, particularly of the chelipeds
and cephalothorax. The degree of hyperpigmentation appears to
develop with increasing infection severity and was a major feature
in the initial discovery of the condition in Scottish N. norvegicus
during the early 1980s. The external appearance of the carapace
has been used as a field diagnostic tool for Hematodinium infection
of N. norvegicus in Scottish (Field et al., 1992; Stentiford et al.,
2001c), Swedish (Tärnlund, 2000) and Irish (Briggs and McAliskey,
2002) waters. In addition, since this symptom of infection is also
observed in other Hematodinium-infected hosts, it has been used
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Fig. 4. Transission electron micrographs of Hematodinium perezi and Hematodinium sp. infecting Nephrops norvegicus, Cancer pagurus, Callinectes sapidus, Carcinus maenas and
Liocarcinus depurator. (A) Bi-nucleate stage of Hematodinium sp. from N. norvegicus containing nuclei with condensed chromatin (black arrow), abundant trichocysts (white
arrow) and vacuoles (v) throughout cytoplasm. Scale = 2 lm. (B). Multi-nucleate plasmodium of Hematodinium sp. from N. norvegicus showing similar features to A.
Scale = 2 lm. Inset: higher power image of cross section of several trichocysts. (C). Multi-nucleate plasmodium of Hematodinium sp. from C. pagurus displaying similar
features to A and also, abundant mitochondria and other organelles within cytoplasm (asterisk). Scale = 2 lm. (D). Apparently uninucleate stage of Hematodinium sp. from C.
sapidus displaying similar features to A, B and C but with clearly visible alveolar membrane, raised from the cell surface in several regions (arrow heads). Scale = 2 lm. (E).
Uninucleate stage of Hematodinium perezi from European C. maenas. Note the apparently different appearance of the cytoplasm of the parasite cell compared to A–D but
presence of a nucleus containing condensed chromatin profiles (arrow) and vacuole-rich cytoplasm (v). Trichocysts were not observed in any of the parasites from this
preparation. Scale = 2 lm. (F). Multi-nucleate plasmodium of H. perezi from European L. depurator. Note the presence of similar features to those depicted in A–D but a
contrast to the ultrastructural appearance of the isolation from C. maenas (E). Scale = 2 lm.
to diagnose Hematodinium sp. in C. pagurus (Stentiford et al., 2002),
C. bairdi (Meyers et al., 1987, 1990) and C. opilio (Taylor and Khan,
1995; Dawe, 2002; Pestal et al., 2003; Shields et al., 2005). However, while this method remains useful for the detection of advanced infections, it does not detect low-level ‘sub-patent’ or
potentially latent (low-level, tissue-based) infections in N. norvegicus (Tärnlund, 2000; Stentiford et al., 2001c).
Recognising the limitation of carapace colouration as an accurate diagnostic tool for Hematodinium infections of N. norvegicus,
Field et al. (1992) developed a novel approach to diagnosis via
visualisation of the haemolymph through the thin cuticle and epithelia of the pleopods. Not only did this technique diagnose infection per se, it also allowed assignment of a grade of relative severity
(Field et al., 1992; Field and Appleton, 1995). A pleopod is removed
from the abdomen and assessed for the presence of parasites within the haemal space using low-power microscopy. This method has
been used in field studies of Hematodinium infections in populations of N.norvegicus from the Scottish west coast (Field et al.,
1992, 1998; Stentiford et al., 2001c) and allows for detection of
4–50% more infected lobsters than the carapace discolouration
method (Tärnlund, 2000; Stentiford et al., 2001c). In addition to
its utility as a diagnostic tool during field surveys, the technique
has also been used to grade infection severity of N. norvegicus during numerous studies of the pathological, physiological and behavioural manifestation of Hematodinium infection in host lobsters. As
such, despite recent advances in antibody and molecular based
diagnostic methodologies, the pleopod staging technique remains
a useful tool for use by field and laboratory scientists.
Several studies have used histological preparations of the haemolymph and tissues for the diagnosis of Hematodinium infection.
Histology offers significant advantages to either carapace or pleopod based diagnosis since it allows for direct visualisation of the
G.D. Stentiford, D.M. Neil / Journal of Invertebrate Pathology 106 (2011) 92–109
pathogen and associated effects within the host. This capacity is
improved further when electron microscopy is employed to provide an ultrastructural perspective. Methanol-fixed haemolymph
smears stained with either Giemsa or haematoxylin and eosin provide satisfactory results (see Meyers et al., 1987; Love et al., 1993;
Hudson and Shields, 1994; Messick, 1994; Taylor and Khan, 1995;
Wilhelm and Mialhe, 1996; Messick and Shields, 2000) though wet
smears, prepared using poly-L-lysine-coated slides, fixed in Bouins
solution or 10% neutral buffered formalin (NBF), and stained with
haematoxylin and eosin procedure or a modified Giemsa provide
a more reliable diagnostic medium (Messick, 1994; Messick and
Shields, 2000; Shields and Squyars, 2000; Pestal et al., 2003).
Non-lethal methods based upon analysis of haemolymph samples
are useful since they allow pathogenesis to be monitored in laboratory settings. However, since evidence exists to suggest that
sub-patent and latent infections may not be visible within haemolymph smears, more accurate diagnostic assessments can be made
using organ and tissue histology. While several fixatives have been
used for histology of crustacean tissues, Davidson’s seawater fixative (Hopwood, 1996) appears to offer consistent results (certainly
for marine decapod species), though we have used 10% NBF to good
effect in animals collected from estuarine habitats. For stenohaline
N. norvegicus, tissue and organ samples (normally heart, hepatopancreas, cheliped muscle, abdominal muscle, gonad and gill) are
excised and fixed in Davidson’s seawater fixative for 24 h before
transfer to 70% industrial methylated spirit for storage prior to processing. Fixed samples are then prepared for histology using standard protocols (for example see Stentiford et al., 2002) and stained
using haemotoxylin and eosin. Hematodinium parasites are easily
diagnosed in sections due to their condensed chromatin profiles
that stain densely with haemotoxylin. For electron microscopy,
small samples (c. 2 mm3) of hepatopancreas should be fixed in
3% glutaraldehyde in 0.1 M sodium citrate buffer (pH 7.4) with
1.75% sodium chloride for 2 h at room temperature followed by
post-fixation in reduced 1% osmium tetroxide for 1 h at 4 °C (for
example see Stentiford et al., 2002). Uranyl acetate staining will
define uninucleate and multi-nucleate stages of Hematodinium
based upon their characteristic nuclei with condensed chromatin
profiles, cytoplasmic trichocysts and a bounding alveolar membrane. At present, ultrastructural diagnosis is considered the definitive diagnostic tool for Hematodinium infections of N. norvegicus.
Stentiford and Shields (2005) note that while Neutral Red is an
excellent vital stain for Hematodinium, at least in fresh smears of
H. perezi in green crabs (Chatton and Poisson, 1931) and infections
in blue crabs (Shields, J.D., unpubl. data), it does not stain Hematodinium from N. norvegicus, and thus is not a good indicator for
Hematodinium infections in general. Janus Green has also been
used as a vital stain (Chatton and Poisson, 1931) but its use as an
indicator of infections has not been evaluated.
Due to the commercial significance of Hematodinium as a pathogen of N. norvegicus, it is perhaps not surprising that considerable
attention has been afforded to the development of more specific
diagnostic tests based upon immunological and molecular technologies. The successful in vitro culture of Hematodinium isolated from
N. norvegicus by (published by Appleton and Vickerman (1998))
was critical in allowing antibodies to be raised against the parasite
and further, for development of an indirect immunofluorescent
antibody technique (IFAT) for diagnosis (Field and Appleton,
1996). The IFAT provided the first clue that apparently uninfected
N. norvegicus could harbour sub-patent or even latent infections
(Field and Appleton, 1996) and that the field analysis techniques
based upon the carapace colour and pleopod technique may be
underestimating true field prevalence. The development of a Western-blot technique using these antibodies allowed for the objective monitoring of Hematodinium infection in the N.norvegicus
fishery over a complete season, providing more accurate data on
101
infection-associated mortality for potential use in stock assessment models (Stentiford et al., 2001d). In this study, it was shown
that the technique demonstrated not only an ability to diagnose
infection earlier in the season but also that the pleopod technique
underestimated prevalence by as much as 25%, particularly in the
early season when infection severity was lower. Using this data,
it was suggested that sensitive immunological diagnostic tools
could be used to predict the patent prevalence that occurs later
in the same season. Whether fisheries managers can utilise the
information to offset mortalities due to development of patent
infection remains to be shown. The technique developed by Field
and Appleton (1996) and utilised by Stentiford et al. (2001d) has
been further developed into an ELISA-based diagnostic test that
provides an even more rapid diagnosis of the disease in this species
(Small et al., 2002). Recent work however has shown that the polyclonal antibody raised against cultured Hematodinium from N. norvegicus can cross react with epitopes found on other protozoan
parasites (Bushek et al., 2002). With this in mind, care must be taken when applying such a technique, particularly where the background pathogen fauna of the host is not well known.
The first PCR-based diagnostic assay developed for the detection of Hematodinium in decapod hosts was reported by Hudson
and Adlard (1994). A 680 bp product was amplified from the 30
end of the SSU region of the 18S ribosomal DNA and a sequence
of this product was subsequently shown to be specific to Hematodinium (Hudson and Adlard, 1996). Comparison of sequences from
several hosts indicated that Hematodinium in C. sapidus was different to that infecting N. norvegicus, C. bairdi and C. opilio. In a later
study, additional primer sets were developed for the Hematodinium
parasite infecting C. sapidus (Gruebl et al., 2002; Sheppard et al.,
2003), these being used to demonstrate sub-patent infections in
blue crabs. In light of concerns about the specificity of the polyclonal antibody raised against cultured Hematodinium from N. norvegicus, Small et al. (2006) have recently reported improved
diagnostic tests for the disease based upon PCR. In this study, Small
et al. (2006) note that the PCR primer sets used in previous studies
of Hematodinium in decapods have been based upon conserved regions of the 18S and 5.8S regions of the rDNA. As such, the authors
state that such primer sets are not specific for particular Hematodinium species. Using approaches based upon amplification of variable regions of the parasite genome, Small et al. (2006) report a
species-specific primer set for Hematodinium infection of N. norvegicus. A diagnostic band at 380 bp allows for the definitive diagnosis of Hematodinium sp. from N. norvegicus and now opens the
potential for sensitive epidemiological investigations of infection
in this species and others that may host the same parasite within
the fishery. Furthermore, the demonstration of parasite labelling
using the in situ hybridization protocol described by Small et al.
(2006) will now allow for parasite transmission and early pathogenesis trials to be carried out. Since transmission is significantly
understudied in this parasite group, this should be seen as a promising new development.
Whilst molecular tools are clearly assisting field scientists with
diagnosis of Hematodinium infection in decapods, the expression
and sequencing of elements of the parasite’s genome (via intelligent
primer design and PCR) is likely to prove fruitful in establishing the
taxonomic link between H. perezi and the Hematodinium-like parasites infecting other crustacean hosts, including N. norvegicus. As
stated above, accurate species descriptions are required in order
to assess the biosecurity risk of decapod movements, particularly
to areas where commercial capture and culture industries exist.
4.4. Current status of infection prevalence monitoring
The defining characteristics of enzootic Hematodinium spp.
infection systems have until now been regarded to be an annual
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and highly seasonal occurrence of patently infected hosts, with a
mortality of epizootic proportions in the infected hosts and a subsequent ‘low’ season when infected hosts are not abundant and are
sometimes undetectable (Briggs and McAliskey, 2002; Chualain
et al., 2009; Field et al., 1992, 1998; Love et al., 1993; Messick and
Shields, 2000; Meyers et al., 1990; Sheppard et al., 2003; Stentiford
et al., 2001c,d; Stentiford and Shields, 2005). The infection system in
the N. norvegicus fishery in Clyde Sea Area in Scotland has exhibited
these characteristics for over 20 years, with the year-on-year peaks
of patent prevalence remaining stable at 20–25% of the sampled
catches (Field et al., 1992, 1998; Stentiford et al., 2001c,d; Beevers,
2010), This seasonal epizootic from an apparently parasite-free
population is a feature that is consistent with the existence of
mechanisms and reservoirs to perpetuate the infection from year
to year. However, these methods are subjective and lack sensitivity,
and thus may have overlooked low-level infection.
More recently, through the use of a sensitive high throughput
enzyme linked immunosorbent assay (ELISA) (Small et al., 2002)
and polymerase chain reaction assays (PCR) (Gruebl et al., 2002;
Small et al., 2006) alongside the subjective measures of patent disease to monitor the presence of infected animals throughout the
infection peaks and nadirs in the Clyde Sea, the limited sensitivity
of the subjective measures of patent disease has been highlighted.
However, use of a multiple assaying technique (using ELISA, PCR,
the pleopod method and the body colour method) has revealed
pre-patent levels of infection comparable to the patent disease
peak throughout the year, and a level of prevalence that, although
fluctuating, is consistently above the values previously reported
during the apparent infection nadirs (Beevers, 2010). These results
therefore challenge the classification of the patent disease peak as
epizootic in nature. For this reason it may be argued that it is not
appropriate to describe the Hematodinium sp. associated disease
in N. norvegicus from the Clyde Sea as a seasonal disease, but rather
that patent disease be described as ‘seasonally apparent’.
By holding in tanks sub-patently Hematodinium sp. infected N.
norvegicus caught in the patent infection nadir, it has been possible
to show that patent infection development can take up to
9 months, with gross signs of the disease being concurrent with
that observed in the wild fishery (Beevers, 2010). These observations provide evidence that at least some of the infected hosts
found in the mortality peaks for N. norvegicus might be represented
by animals that were infected the previous year or earlier. However this does not exclude the possibility that alternative hosts exist in the life cycle of these parasites or that infection and death
due to advanced disease happen in the same year.
Seasonal peaks in patent Hematodinium disease have also been
reported in other wild, commercially-harvested decapods (Stentiford and Shields, 2005), and it is possible that in these cases also
infected hosts are present throughout the year, though not detected either due to limitations in the sampling methodology or
a low sensitivity of the assays used. The application of more sensitive diagnostic methods to these infection systems should therefore be a priority for future research. One current approach to
such a thorough and ongoing monitoring of parasite spread and
load in fisheries and associated marine fauna is using parasite
material to generate both PCR-based diagnostic tools, capable of
discerning species and strain differences of parasites, and antibody-based dipstick tests that can be employed in the field without
specialist training (Gornik, S., pers. comm.). Candidate genes/proteins for antibody generation can be identified using a molecular
data set of highly-expressed proteins specific to the infection stage
of Hematodinium using (1) analysis of expressed genes as mRNAs
from EST data, and (2) protein data analysed by two-dimensional
protein electrophoresis and mass-spectrometry. ELISA and lateral
flow-type diagnostic tools can then be developed using antibodies
raised against such highly expressed parasite proteins.
4.5. Pathogenesis
A section on the pathogenesis of any disease should rightfully
start by considering the mode of transmission of the parasite between susceptible hosts. However, despite a considerable body of
knowledge on this parasite and its existence in wild populations
and significant advances in our ability to diagnose infection (even
before disease manifests), very little is known about its transmission and of the early stages of infection. This is likely a reflection
of the difficulty in defining pathogenic processes in wild marine
populations but also indicates a knowledge gap in the taxonomy,
life cycle, host range and latency periods for this parasite group,
particularly in relation to the discontinuous life history patterns
of their hosts.
Hematodinium infections have been transmitted via inoculation
of infected haemolymph into naïve C. bairdi (Meyers et al., 1987), C.
sapidus (Shields and Squyars, 2000) and Portunus pelagicus (Hudson
and Shields, 1994). These trials have demonstrated that filamentous trophonts and vegetative, amoeboid trophonts can establish
infection (Meyers et al., 1987; Hudson and Shields, 1994; Shields
and Squyars, 2000). Previously, micro- and macrospores were also
shown to produce infections when inoculated into C. bairdi (Eaton
et al., 1991) though this could not be demonstrated using a similar
approach in N. norvegicus (Appleton and Vickerman, 1998). Stentiford and Shields (2005) have previously stated that despite its
simultaneous production and active en masse exit from heavily infected hosts, the dinospore is not necessarily the infective stage but
instead an intermediate stage preceding a resting cyst or some
other non-parasitic stage (see Shields, 1994). This theory is partially supported by the report by Appleton and Vickerman (1998)
that the culture of dinospores leads directly to the development
of filamentous trophonts in vitro. While in vitro sporulation of
Hematodinium from N. norvegicus is not synchronous, the event
within infected host lobsters appears to be so with almost complete exsporulation from host gills and arthrodial membranes
and death occurring soon after (Appleton and Vickerman, 1998).
Similar events have been recorded in C. pagurus (Stentiford and
Shields, 2005) though in C. sapidus at least, there is potential for
multiple sporulation events within a given host, some of which
may lead to high spore densities and some of which do not (Shields
and Squyars, 2000).
Despite the identity of the infective stage(s) within the life cycle
of the parasite, some uncertainty also exists surrounding the route
of entry into naïve hosts. Several studies have suggested that the
moult stage is an important period for obtaining infection but data
is circumstantial, particularly considering the apparently extended
incubation period to patent disease and the difficulty in relating an
infection event to a mortality event or season (Stentiford and
Shields, 2005). There is evidence for transmission to naïve C. sapidus via ingestion of infected food (Sheppard et al., 2003), though
this was not the case for P. pelagicus (Hudson and Shields, 1994).
Given the potential cannibalistic tendencies and scavenging nature
of many decapod crustacean species, this route for transmission
appears likely (Sheppard et al., 2003). Further possibilities, at least
in the case of N. norvegicus are that dinospores are ingested via suspension feeding (which occurs during certain seasons) (Loo et al.,
1993), or that intermediate hosts, such as the benthic amphipod
Orchomene nanus (a scavenger with preference for crustacean carrion) are intermediate or reservoir hosts for the parasite (Johnson,
1986; Stentiford and Shields, 2005; Small et al., 2006). Some studies have suggested potential for sexual transmission (Meyers et al.,
1996) though this appears somewhat unlikely given the arrested
development and significant pathological disruption of the ovary
in infected female hosts (see Stentiford et al., 2002). Clearly, dedicated studies on the transmission of Hematodinium between infected and naïve N. norvegicus are required to clarify this issue
G.D. Stentiford, D.M. Neil / Journal of Invertebrate Pathology 106 (2011) 92–109
and to ascertain whether commercial practices (such as the discarding of disassembled catch) may contribute to the spread of this
disease in the fishery. The use of emerging molecular tools, particularly PCR and ISH should assist these studies.
Field et al. (1992) provided the first description of the pathogenesis of Hematodinium in N. norvegicus, following studies of the
Scottish west coast fishery. The pathological manifestation of the
disease in decapods has been amply covered by several authors
and is also described in detail elsewhere in this volume. A brief
overview is given here. Due to the relatively open circulatory system of decapods and the systemic nature of Hematodinium infections, it is not surprising that all major organ systems are
variously impacted during disease. Externally, patently infected
lobsters display a brightly coloured but opaque (‘cooked’ appearance) carapace that remains following haemolymph removal and
death of the host. Haemolymph drawn from patently infected lobsters is milky white with increased viscosity and opacity. Centrifugation of haemolymph from lobsters in advanced stages of
infection can produce cell pellets amounting to approximately
50% of the sample by volume (Stentiford, G.D., pers. obs.), equating
to the approximate 8-fold increase in circulating cells compared to
uninfected lobsters reported by Field et al. (1992). The remaining
plasma fraction is generally devoid of colour and has a significantly
increased clotting time and often fails to form a normal clot. The
haemolymph cell fraction from the majority of heavily infected
lobsters consists of non-motile stages similar in size to host haemocytes (5–14 lm in diameter). Most of these are uninucleate
while others contain multiple nuclei. Field and Appleton (1995) report that four stages are commonly observed within infected N.
norvegicus – these being uninucleate forms and multinucleate plasmodia circulating freely in the haemoymph, filamentous syncytia
attached to host organs and a separate syncytial network ramifying
between (for instance) muscle fibres of the abdomen and heart.
These forms approximately correlate to the developmental stages
103
described in vitro by Appleton and Vickerman (1998). Furthermore,
infected lobsters containing masses of bi-flagellate stages corresponding to the macrospore and microspore forms (Field and
Appleton, 1995; Appleton and Vickerman, 1998) are occasionally
encountered. The uninucleate stages encountered free within the
haemolymph likely correspond to the sporoblast stages described
by Appleton and Vickerman (1998) and it is these that give rise
to the flagellated spore stages encountered in some animals. Presumably the multinucleate syncytial networks (either attached to
the surface of host organs or ramifying between three dimensional
structures such as the abdominal musculature) give rise to the circulating stages and it is via these attached stages that infection appears to establish within the host. This is reinforced by reports on
the detection of parasites (via immunological and molecular diagnostics) within organs prior to their appearance in the haemolymph (Field and Appleton, 1996; Stentiford et al., 2001d; Small
et al., 2002). These attached stages also compose the ‘creamy
deposits’ reported to occur coating the internal organs of Hematodinium-infected hosts (Stentiford and Shields, 2005) (see Fig. 5).
It is generally reported that crabs and lobsters infected with
Hematodinium exhibit haemocytopoenia (reduction in the haemocyte count) though this has only been demonstrated quantitatively
for infected C. sapidus (Shields and Squyars, 2000). Studies on N.
norvegicus have demonstrated that the combined cell count for
haemocytes and parasites significantly increases during patent
infections though specific counts on haemocytes were not carried
out (Field and Appleton, 1995). Despite significant declines in haemocyte counts reported for C. sapidus, other studies have shown
that although high parasite numbers are recorded during patent
Hematodinium infections, efficient haemocyte responses can occur
to other pathogens that may co-infect the host. In the case of C.
pagurus, co-infecting yeast pathogens elicited a pronounced immunological response from crabs displaying advanced Hematodinium
infections, suggesting that although circulating haemocyte
Fig. 5. Hematodinium sp. infestation of the ovary of N. norvegicus. (A). Ovarian follicle infiltrated by large numbers of uni- and multi-nucleate stages. Oocytes are apparently
arrested in a pre-vitellogenic state (arrow). Bar = 200 lm. (B). Multinucleate plasmodial stages attached to the surface of a degenerate oocyte (arrow). Bar = 50 lm. (C).
Necrotic vitellogenic oocytes containing remnant vitellogenin inclusions (arrow) or a fine granular matrix (asterisk). Bar = 100 lm. (D). Droplets of vitellogenin (arrow)
apparently liberated from necrotic vitellogenic oocytes, among parasite plasmodial stages. Bar = 50 lm. Material used for obtaining images courtesy of Dr. Beth Leslie,
Atlantic Fisheries College, Shetland.
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numbers may be apparently reduced, the host can still respond to
pathogenic challenge when stimulated appropriately (Stentiford
et al., 2002). Field and Appleton (1995) demonstrated apparent
stimulus of haemopoietic activity in heavily infected lobsters
though this did not appear to manifest as increased numbers of released or circulating haemocytes. Evidence from the literature suggests a rather complex immunological relationship between
Hematodinium and its decapod hosts and further investigations
on the interaction between hosts and their parasite isolates may
provide intriguing insights into the pathogenesis of this disease.
4.6. Pathology and its effect on commercial products
In keeping with the scope of this volume, it is important to consider the pathological manifestations of Hematodinium infection,
particularly in light of their effect on the commercial exploitation
of N. norvegicus as fisheries products. Since the late 1990s, a high unit
value has designated N. norvegicus as the most valuable fisheries species landed in the UK and Ireland. Product is sold live, fresh dead or
frozen mainly from the UK, Denmark and Ireland with major exportation to Italy, France and Spain, and to countries outside of the European Union. The live market is largely dependent on trap-caught
animals, while trawl fishery dominates the fresh and frozen markets
(Bell et al., 2006). Health and quality of product is clearly an important consideration for the live market (where animals are shipped to
market destinations via road in ‘vivier’ systems). For the frozen market, animals are often ‘tailed’ at sea, the abdominal section being processed for the production of ‘scampi’. In general, smaller animals are
captured via trawling and larger animals by trapping. The abdominal
musculature is the major harvestable product in both cases.
Several studies have considered the effect of Hematodinium on
the abdominal musculature of N. norvegicus. Field and Appleton
(1995) state that despite the presence of network-like parasite syncytia between muscle fibres and the association of uninucleate
forms with the sarcolemmal membrane, histology of the abdominal musculature remained largely normal, even in advanced stages
of infection. Later studies demonstrated that the water content of
the abdominal musculature was relatively unaltered in late stage
infections and that ultrastructural damage to the muscle was generally limited to the fibre periphery (Stentiford et al., 2000b). The
effect of Hematodinium infection on the abdominal musculature
of N. norvegicus contrasts that seen in some other Hematodiniumhost models. In several crab species, the gross appearance, water
content, histology, mechanical structure and texture of the muscle
is altered during late stage infections (Meyers et al., 1987; Field
et al., 1992; Messick, 1994; Hudson, 1995; Wilhelm and Mialhe,
1996; Stentiford et al., 2002; Stentiford and Shields, 2005). This
is perhaps best demonstrated in C. pagurus where Hematodinium
infections cause almost complete degeneration of the claw musculature with separation of the sarcolemma from contractile myofibrils and severe disorganisation of filaments in the region of the
Z-line (Stentiford et al., 2002). Unpublished results from our laboratories have shown that in contrast to the abdominal musculature, the histological structure of the cheliped muscles of
infected N. norvegicus is significantly altered similar to that seen
in infected crabs, even during early stage infections. The contrasting effects of the disease on different muscle groups of N. norvegicus and between N. norvegicus and other decapods has previously
been discussed in relation to differential responses of these muscle
groups to parasite-derived proteases or to the specific structural
properties of the abdominal musculature of N. norvegicus that prevents severe infiltration or collection of the parasite in this body region (see Stentiford et al., 2000b, 2002; Stentiford and Shields,
2005). Whatever the cause, the relative lack of effect of disease
on the abdominal musculature is perhaps fortuitous for the fresh
dead and frozen ‘tailed’ market for N. norvegicus since at least in
terms of mechanical integrity, the product appears not to be
greatly altered.
Despite the general lack of effect of Hematodinium on the structural integrity of N. norvegicus abdominal musculature, several
studies have demonstrated that significant alterations in host
physiology and biochemistry accompany the progression of this
disease. The massive proliferation of parasite stages within the
haemolymph of infected lobsters (and crabs) lead to a pronounced
hypoxia and to a switch to anaerobic metabolism during the advanced stages of the disease (Taylor et al., 1996; Love et al.,
1996; Shields et al., 2003). Infected lobsters display perturbations
in carbohydrate handling capacity with reserves of glycogen in
the muscle and hepatopancreas being significantly reduced concomitant with infection (Stentiford et al., 2000b, 2001a). Glycogen
from these regions is converted to glucose in response to stress
(e.g. hypoxia). The response is controlled by the release of crustacean hyperglycemic hormone (CHH) from the sinus gland. In infected lobsters, plasma concentrations of CHH are significantly
elevated, probably due to a parasite-derived disruption in feedback
loops that control the release of the hormone from the sinus gland
(Stentiford et al., 2001a). With advancing disease status, the elevated concentration of plasma CHH causes a depletion of glycogen
in the hepatopancreas and a transitory increase in plasma glucose
(presumably utilised by the developing parasite population) (Stentiford et al., 2001a). Hematodinium infection also results in a complete depletion of glycogen in the abdominal muscles (Gornik et al.,
2010) Reductions in glycogen have also been reported to occur in
several other Hematodinium-decapod models and likely represent
a causal factor for morbidity and eventual mortality in infected
hosts (Stentiford et al., 2000b; Shields et al., 2003). In addition to
disruptions in carbohydrate profiles, detailed studies on the free
amino acid (FAA) profiles of plasma and muscle have also been carried out on Hematodinium-infected lobsters (Stentiford et al., 1999,
2000b) and on the ratio of nucleotides in the muscle, as expressed
in the adenylate energy charge (AEC) (Gornik et al., 2010). Tissue
and haemocyte degradation and the induction of a generalised host
stress response were associated with an increase in the concentration of FAAs such as taurine during infection. Also, the muscle AEC
values were significantly reduced compared with those of uninfected animals. Such changes, in addition to those recorded for proteins (above) and salts in the plasma of Hematodinium-infected
crabs (Hudson, 1995; Love et al., 1996) indicate a departure from
the normal electrolyte status during disease and suggest induction
of a significant stress cascade in infected hosts. As expected, the effect of alterations in salt, glycogen, FAA, nucleotides and protein
handling in infected hosts are reflected in the organoleptic qualities of the abdominal musculature of infected animals. In a trial
with a trained sensory panel, considerable differences have been
detected between samples of fresh tail meat from uninfected and
from heavily-parasitised animals with the latter being described
as bland in flavour and after-taste, slightly less firm and less chewy, and with an overall lower liking (Neil et al., unpubl. data) In
addition to alterations in the palatability of meat harvested from
infected lobsters destined for the frozen and fresh dead market,
departures from the normal physiological and biochemical wellbeing of the host will also impact upon survival and quality of animals destined for the live market. The impact of diseases such as
Hematodinium as drivers for post-capture mortality events in decapods destined for live markets is currently understudied. Since
other organ and tissue systems are not considered as direct commercial products from N. norvegicus, detailed descriptions of the
pathology of these organs has not been provided here. The subject
matter is described in more detail by Field and Appleton (1995)
and by Stentiford and Shields (2005). Furthermore, Morado covers
the pathological manifestation of Hematodinium infection in crabs
within this volume.
G.D. Stentiford, D.M. Neil / Journal of Invertebrate Pathology 106 (2011) 92–109
4.7. Hematodinium changes host behaviour
Based on the physiological perturbations imposed on lobsters
by patent Hematodinium infection, several studies have considered
how these effects may alter the locomotory performance and life
history traits of host lobsters. N. norvegicus populations are generally found burrowing into soft muddy sediments and their capture
(by trawlers) depends on emergence from the burrow and an
inability to escape (via tail flipping) from an advancing fishing
net (Farmer, 1974c). Since lobsters predominantly emerge to feed,
any factor that alters the requirement for food may be expected to
affect this entrained diurnal emergence behaviour (Farmer, 1974a).
Furthermore, once out of the burrow, any factor that impedes
swimming behaviour may also impact upon catchability by predators and the fishery (Newland et al., 1992). Both the speed and
endurance of tail flip swimming have implications for capture by
trawl nets. Studies by Stentiford and colleagues have demonstrated
how burrow emergence pattern and swimming endurance are significantly altered during patent Hematodinium infections in N. norvegicus. In controlled tank trials using time-lapse filming of burrow
emergence behaviour of healthy and diseased lobsters, heavily infected lobsters were spent more than 10 times longer out of the
burrow than their uninfected counterparts. Infected lobsters also
showed a loss of the diurnal (dusk/dawn) emergence pattern observed in uninfected lobsters. The increased time spent out of the
burrow was interpreted as a symptom of ‘physiological starvation’
whereby the progressive consumption of host nutrient reserves by
the growing parasite population necessitated increased feeding
activity by infected animals. Alternatively, relatively hypoxic conditions within the burrow may have forced emergence onto the
sediment surface in disease-stressed lobsters (Stentiford et al.,
2001b). One intriguing event, reported by Stentiford et al.
(2001b), was the spasmodic tail flipping that occurred in two lobsters immediately prior to death during filming. Whether this
event coincided with the exsporulation phase of the disease was
not evident from the recordings though it is interesting to speculate that some active manipulation of host behaviour by the parasite population may assist with liberation of dinospores from
terminally infected lobsters.
In addition to the increased time spent out of the burrow, infected lobsters also exhibit significantly reduced swimming ability.
Hematodinium-infected lobsters showed a reduction in the number
of tail flips performed, the number of swimming ‘bouts’ performed
and the total distance travelled, concomitant with increasing disease burden. Further, the velocity of the first (giant-fibre mediated)
flip was significantly less in diseased animals (Stentiford et al.,
2000a). Studies on burrowing behaviour and swimming performance in diseased lobsters demonstrate how parasites may affect
key life history traits of decapods. In addition to contributing towards the efficient transmission of parasites between hosts, some
changes may also affect the availability of infected animals to the
fishery (Field et al., 1998) and should be considered when estimating prevalence and mortality in natural populations (Stentiford and
Shields, 2005).
4.8. Hematodinium epizootiology
Studying parasite epizootiology in wild decapod populations is
often hampered by inconsistent access by scientists to the fishery
and an absence of long-term monitoring programs for commercially significant pathogens. The importance of Hematodinium as
a mortality driver in the Scottish N. norvegicus fishery has been
recognised since the early 1990s and considerable effort has been
devoted to understanding the life history of this parasite in Scottish
and Irish waters. Similar recognition of the importance of this parasite in temperate water crab stocks has led to Hematodinium being
105
the perhaps the best studied parasite of commercially exploited
wild decapod stocks. Comprehensive surveys of Hematodinium
prevalence in Scottish and Irish N. norvegicus populations using
the pleopod diagnostic technique have demonstrated a consistently expressed seasonality with maximal prevalence reaching
up to 70% during the spring (February–April) and lowest levels of
detectable disease during the summer and autumn (July–October)
(Field et al., 1992, 1998; Field and Appleton, 1995; Stentiford et al.,
2001c,d; Briggs and McAliskey, 2002). More detailed surveys that
attempted to include latently and sub-patently infected animals
into prevalence estimates using antibody-based diagnostics demonstrated considerable underestimation of prevalence (when using
the pleopod method) in the early season, with this decreasing as
infections progress to patent disease in the later season (Stentiford
et al., 2001d). Recent developments in molecular diagnostic tools
for Hematodinium may allow this issue to be re-addressed, utilising
the increased sensitivity of nucleic acid detection over the antibody techniques employed by Stentiford et al. (2001d) (Small
et al., 2006, 2007). By combining in situ hybridization technologies
with controlled transmission studies researchers should be able to
identify initial infection sites on the host and further to elucidate
early events in the pathogenesis of disease.
When studying the epizootiology of Hematodinium infection in
decapods, it is important to consider information on potential reservoirs or alternative hosts to the pathogen and how infection
prevalence in these organisms may affect that seen in the target
fishery. In studies off the north east coast of the United States,
Johnson (1986) demonstrated the presence of dinoflagellate parasites of the Order Duboscquodinida, family Syndinidae in several
species of benthic amphipods. The parasites were morphologically
most similar to the decapod pathogen H. perezi. As in decapods,
host reaction to the presence of parasites was very rare. In some
cases, a co-infection with an unidentified fungus elicited a strong
host reaction suggesting that host immune ability was not degraded by Hematodinium infection but rather that Hematodinium
evades the system, a feature also recognised by Stentiford et al.
(2003) for Hematodinium-yeast co-infections of the crabs C. pagurus and N. puber.
Similar studies have been carried out on the copepods. Calanus
finmarchicus collected from the Clyde Sea Area in Scotland (the
main site for the majority of studies on Hematodinium infection
in Scottish N. norvegicus described above) were host to several
dinoflagellate parasites (including Syndinium sp. and several more
haemocoelic pathogens of ‘more doubtful status’) (Jepps, 1937) and
high mortalities of Paracalanus indicus due to dinoflagellate parasites were reported by Kimmerer and McKinnon (1990). Considering these studies and the apparent propensity for non-decapod
hosts to harbour infections by Hematodinium-like pathogens, it is
tempting to suggest that these hosts may play an important role
in the life history of Hematodinium infections of commercially significant decapods. Using molecular diagnostic and pathogen typing
tools and by considering diseases of commercial species at the level of the ecosystem in which these hosts exist, it should now be
possible to better investigate the life cycles of these important
pathogens and to understand the interactions between apparent
definitive and alternative or reservoir hosts. Studies of this kind
may also allow for interpretation of the seasonal peak-nadir pattern of Hematodinium infections in commercially exploited decapods by identifying the ecosystem compartment where the
pathogen resides when outside of the target host.
4.9. Direct and indirect mortality
Consensus opinion of those carrying out studies on Hematodinium
and Hematodinium-like dinoflaglellate infections of decapods considers that patent disease has a fatal outcome with little evidence
106
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for recovery (see Stentiford and Shields, 2005) and this is particularly
apparent for patently infected N. norvegicus (Field et al., 1992).
Assessment of long-term field data for N. novegicus burrow counts
(an accepted indicator of stock density) has shown that reductions
in burrow count and landings per unit effort (LPUE) coincided with
the highest prevalence of Hematodinium in N. norvegicus from this
fishery (Field et al., 1998). While it is not possible to solely ascribe
this reduction to the parasite, the data highlights how disease may
contribute a significantly higher proportion of natural mortality than
traditionally allowed for in fisheries models. Interestingly, several
studies have also demonstrated how Hematodinium prevalence is
highest at sites where N. norvegicus populations are smallest and/
or are composed of relatively size matched individuals (Field et al.,
1998; Stentiford et al., 2001c). Since population size-structuring is
likely to be altered by fishing pressure, it is intriguing to propose that
such anthropogenic drivers for population restructuring may have
an effect on the detected prevalence of pathogens within hosts from
those populations. In such a way, where diseases are deemed to have
‘emerged’ within a fishery (as apparent for Hematodinium in N. norvegicus populations from the Clyde Sea Area, Scotland in the late
1980s), emergence may be based on movement towards a higher
proportion of susceptible hosts within the population (i.e. within a
given size range or age) rather than a sudden environmental change
or appearance of a pathogen. Given this effect, management of
Hematodinium epidemics within N. norvegicus populations may be
better predicted (and potentially mitigated) by closer observation
to size-structuring in host populations and management of fisheries
effort in populations that appear to be reaching optimal structuring
for epidemics to occur.
In addition to direct mortality effects of Hematodinium in N. norvegicus, evidence also exists for effective castration of individuals
and host populations. Shields (1994) notes that while dinoflagellate
infections of crustaceans are typically parasitic castrators, castration
has not been examined in crabs or lobsters infected by Hematodinium.
With this said, several authors have noted severe infiltration of the
ovary of infected crabs by Hematodinium (Messick and Shields,
2000; Stentiford et al., 2002) with apparent cessation of oocyte maturation in infected females. In this way, castration could be effected
through the disruption of the testis or ovary of infected hosts. Briggs
and McAliskey (2002) have demonstrated that infected female
N. norvegicus from the Irish Sea do not develop mature gonads and
we have shown that the gonads of N. norvegicus are destroyed during
patent infections (Fig 5). Since size at maturity and peak size for
Hematodinium infection prevalence coincides at approximately
21–34 mm in female N. norvegicus (Hillis and Tully, 1993; Tuck
et al., 2000), the potential for disruption to recruitment in affected
populations appears to be high. Further consideration of this issue
is recommended in future studies.
5. Conclusions and future directions
Despite the relatively replete literature on the effects of Hematodinium infection on N. norvegicus, there is a comparative dearth
of information on other pathogens (and their effects) of the clawed
lobster genera Nephrops and Metanephrops. A lack of field surveillance driven by a perceived inability to manage and control disease
in commercially exploited lobsters (and other wild crustaceans) is
likely the reason for this. The outcome is a literature that appears
to fall short on the information required to properly manage this
important commercial resource and one that fails to fully assess
the effect that disease may have on regulation of stock size and
structure. Furthermore, attempts to assess the effect of diseases
(such as Hematodinium) on the fishery stock of lobsters is hampered by a lack of coherence between data sets related to disease
prevalence and those pertaining to stock size and structure. Future
efforts to integrate data collection procedures for fisheries and disease will lead to a better understanding of natural mortality in the
field and may allow managers to more accurately discriminate
sites that are more heavily affected by disease from others that
are not. In addition, closer surveillance of wild populations will
provide early warning for impending epidemics based around
anthropogenically derived shifts in the population size, age and
sex structure caused by fishing pressure. An increasing reliance
on crustacean fisheries, particularly in the European marine area
where Nephrops is considered a key resource will necessitate and
drive this process.
Several research areas are particularly lacking. Firstly, none of
the studies described in this chapter have considered the presence
and effect of diseases in juvenile lobsters. As stated elsewhere in
this volume, in aquaculture scenarios, diseases of juvenile Penaeid
shrimp have played a highly significant role in defining success
during on-growing phases of production. While diseases are often
visible in the exploited (adult) portion of the fishery, they may
potentially play an even more important role in defining the success of earlier life stages, and therefore in overall recruitment success for the fishery. The Nephrops fishery potentially provides an
excellent model for this type of research since adults and juveniles
are found on the same fishing grounds (and even share the same
burrows during early development) and to a certain extent can
be collected with similar fishing technologies. In other lobster genera (such as Homarus) juveniles are often more cryptic and are
potentially separated from the fishing grounds where the adults
are found. While studying juveniles and adults from the same
grounds will inform on the susceptibility of different life stages
to particular diseases, it will also highlight potentially undiscovered pathogens in host life stages not previously considered. Such
approaches may elucidate immunological reasons for the apparent
lack of description of viral and other pathogens in adult clawed
lobsters. This approach is also consistent with any stock assessment techniques based upon cohort-to-cohort success by providing detailed overviews of the specific pathogen profile of animals
within respective cohorts.
A second major theme for future studies should involve the
effect that commercial practices may have on assisting the perpetuation and spread of pathogens of commercially important crustaceans. A much-neglected potential threat to biosecurity,
particularly within the European Union is the relatively free
movement of live crustaceans from point of capture to point of
market. In several countries (particularly the UK), following capture, wild marine crustaceans (e.g. N. norvegicus, C. pagurus, H.
gammarus) are transported live to continental Europe for resale
and consumption. With the exception of H. gammarus (which
are subject to certain movement restrictions associated with the
notifiable pathogen causing Gaffkemia), the movement of live
crustaceans in this way is relatively uncontrolled with losses in
transport remaining unrecorded and morbid or dead animals
potentially finding their way back into the aquatic environment
at the distant site. Furthermore, water used for transporting animals may be released to local waterways or drains. The significant
dearth in knowledge of potential pathogens of our major commercially exploited species and their potential for transmission to
other commercially exploited and reservoir species identify these
as high risk practices. Live transport of animals to distant markets
when coupled with the apparent high potential for parasites such
as Hematodinium to infect multiple decapod and non-decapod
hosts is cause for concern (Stentiford and Shields, 2005), especially since similar scenarios have previously caused major problems for Penaeid shrimp culture (see Lightner paper in this
volume).
A final theme for future study, particularly where expensive
monitoring programs are devoted to surveying of wild stocks in-
G.D. Stentiford, D.M. Neil / Journal of Invertebrate Pathology 106 (2011) 92–109
volves a shift from screening for individual pathogens of concern at
the time to a more holistic screening program using tools to identify both target and non-target pathogens. Issues such as climate
change are likely to progressively and unexpectedly alter the balance between host, pathogen, and environment. In such a way,
low prevalence of innocuous commensals of today may become
highly prevalent and significant pathogens of the future and alternatively, major pathogens of today may become less of a challenge
for future stocks. Closer attention to pathogens in these wild resources, using holistic tools such as histopathology and dedicated
multiplex molecular diagnostic tools not only inform on likely
driving forces for mortality in wild fisheries but also pre-warn
about potential problems that may exist when attempting to intensively culture these species in the future.
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